Protein-protein interactions play a central role in physiology. In cells, proteins are organized in a complicated and flexible network that can be regulated to ensure almost all cellular processes. When drugs are targeting proteins, they in fact affect the entire networks the targeted protein belongs to, having effects on protein-protein interactions. The direct analysis of the effect of drug candidates on protein-protein interactions or protein complexes is extremely important during the pre-clinical stage of drug discovery when hundreds of potential “lead compounds” must be investigated.
Drug candidates may take the form of small molecules (typically with MW<1000 Da), recombinant proteins, recombinant peptides, antibodies or antibody fragments. After screening of a drug candidate library, the in-depth characterization of the potential therapeutic molecules is crucial for the pre-clinical selection process of lead compounds. This selection process ensures that the best candidates enter the costly clinical process and that the bad or useless candidates are rejected as early as possible in the drug discovery process.
Conventional technologies for characterizing interactions between drugs and proteins include ELISA (Enzyme Linked Immunosorbant Assay) type assays, radioimmunoassay and related techniques and Surface Plasmon Resonance technology (SPR). Among these technologies, SPR is the only one able to characterize “in depth” the interaction between drugs and proteins by measuring binding kinetics, dissociation and association constants and the nature of a target-ligand interaction.
Mass spectrometry is a standard tool for the analysis of proteins after the introduction of the so-called “soft ionization” method, including Electrospray Ionization (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI). These ionization methods have been developed to analyze large molecular weight biopolymers (Fenn et al., Science 246:64-71, 1989; Karas and Hillenkamp, Anal. Chem. 60:2299-3001, 1988). Although mass spectrometry is a standard tool for the analysis of proteins, it is still challenging to use this technology for the analysis of protein-protein interactions and protein complexes. The main difficulty is the tendency of non-covalent interactions between proteins to dissociate during the analysis.
Electrospray ionization (ESI) is the preferred method for the analysis of intact protein complexes as the sample can be analyzed in the presence of favorable buffers, maintaining the interactions stable (Loo, Int. J. Mass Spectrom. 200(1):175-186, 2000). Electrospray ionization mass spectrometry has been used for the analysis of the complex formed between drug candidates Sch 54292, Sch 54341 and Sch 53721 when incubated with the protein ras-GDP (Pramanik et al., J. Mass Spectrom. 33:911-920, 1998). This ionization method has also been used for the analysis of the complexes formed between DNA and small molecules (nogalamycin, hedamycin and distamycin) (Beck et al., Mass Spectrom. Rev. 20:61-87, 2001). Sanglier S. et al., Eur. J. Biochem. 271:4958-4967, 2004 describe the use of ESI-MS for the measurement of ternary complexes resulting from the retinoid corepressor nuclear receptors box peptides interaction with the ligand binding domain of the retinoic acid/retinoid X receptor heterodimer. Finding the favourable conditions to observe intact ions from protein complexes using electrospray ionization is time consuming and still a major difficulty. The major issue when using ESI-MS for determining equilibrium association of non covalent complexes is the difference of response factors for these complexes during ESI ionization that can be due to the collisional activation in the source of the mass spectrometer. Discrimination processes (e.g. mass-dependent ionization efficiency, mass-dependent ion transmission through the mass spectrometer, and non-uniform response of the detector) do not generally allow relating the ion intensities of different species to their solution concentrations. These properties of ESI ionization have major consequences in the case of competition experiments as the response factor for the complexes interacting with different drugs is different (Gabelica, V. et al. J. Mass Spectrom. 38: 491-501, 2003).
Only few studies have been reported for the analysis of intact protein complexes using MALDI ToF mass spectrometry. The main reasons are that non-covalent complexes can dissociate easily not only during the ionization process with laser desorption but also during sample preparation (MALDI-MS; reviewed in Nordhoff et al., Mass Spectrom. Rev. 15:67-138, 1997). Another issue is the ability of a standard MALDI mass spectrometer to detect the intact high-mass protein complexes as MALDI generates mostly single charged pseudo molecular ions. MALDI mass spectrometry has been used for the analysis of intact non-covalent protein complexes using a combination of high-mass detection and cross-linking chemistry (Nazabal, A. et al., Anal Chem. 78:3562-3570, 2006). This analytical method has never been applied to the analysis of drugs targeting protein-protein interactions and protein complexes. MALDI cumulates several disadvantages for this analysis: 1) The laser used for the ionization disrupts the targeted protein complex; 2) The detection sensitivity is reduced or inexistent in the high-mass range; 3) The presence of small molecules in the sample is reducing the ability to detect the high-molecular weight macromolecules because of selective ionization phenomenon; 4) MALDI mass spectrometry is not considered a quantitative tool. There is no direct correlation between the intensity of a peak detected and the amount of protein complexes in a sample.
In WO 2006/116893 (Eidgenössische Technische Hochschule Zürich) a mass spectrometric method is proposed wherein intact ions of undigested, unfragmented covalently stabilized supramolecular target-ligand complexes are analysed with matrix assisted laser desorption ionisation (MALDI). The method is illustrated by the analysis of antibody-antigen complexes and other protein-protein complexes such as complexes between CDC42 and Salmonella outer protein SopE but has never been used for the quantitative analysis of drug candidates targeting protein complexes.